Profile recognition method and apparatus for identifying bacteria

ABSTRACT

A suspension of an unknown micro-organism is introduced into a plurality of reaction chambers, each containing a different specific biochemical reagent. The presence or absence of each reaction is subsequently transformed to a multi-digit numerical profile, which serves to identify the micro-organism by means of a numerical Profile Register. Apparatus is also provided to facilitate the transformation of the test result data to the numerical profile while simultaneously providing a verification of the transformation.

This is a continuation-in-part of application Ser. No. 349,698, filedApr. 10, 1973, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the identification of unknown bacteria bymeans of simultaneously performed biochemical reactions. Moreparticularly it relates to a unique method for transforming the resultsof the biochemical reactions into a numerical profile representative ofthe bacterium being tested and subsequently consulting a ProfileRegister to obtain the identification.

A mechanical coder is provided which enables the user to efficientlytransform the above-stated results to the numerical profile. Thismechanical coder may also be provided with a color check feature toenable the user to visually verify his results.

The identification of bacteria is based on several characteristics ofthese organisms: reaction to certain biochemicals, serological behavior,lysotyping, bacteriophage, morphology, physiology, cell arrangement,etc.

In the routine clinical laboratory, the biochemical reactions form theprimary basis for classification at the level of the tribe and the genuswithin the family Enterobacteriaceae. The behavior of bacterialorganisms with particular biochemicals has been reported by severalinvestigators. See, e.g., Edwards, P. R. and Ewing, W. H. Identificationof Enterobacteriaceae. Third edition. Burgess Publishing Company,Minneapolis, Minnesota., 1972; Le Minor, L. Le diagnostic de laboratoiredes bacilles a gram negatifs Enterobacteries. Tome 1, 4^(e) edition,1972. Editions de la Tourelle, St. Mande - 94, France; Cowan, S. T. andSteel, K. J. Manual for the Identification of medical bacteria.Cambridge at the University Press, 1970; Kauffman, F. The bacteriologyof Enterobacteriaceae. Second edition, 1969. The Williams and WilkinsCompany, Baltimore, Maryland.

Some of these reactions are clear-cut, being mostly positive ornegative, whereas some others are variable. Based on a large number oforganisms, percentages of positive or negative reactions have beenestablished and reported. Several schema to interpret the results ofbiochemical reactions have been developed.

Because of the complexity of the interpretation of the data obtained, asequential method of dichotomous keys, presented in the form of flowdiagrams, is presently widely used. These flow diagrams base theselection of each succeeding biochemical test on the results of theprevious one. This approach, even though practical in routine, is asimplification which, in the case of less common biotypes, can lead tomisidentification.

The possible application of computer technology in the clinicallaboratory has allowed a new approach toward the identification of anorganism in testing it simultaneously with a large number ofbiochemicals. The computer memory can store probability of occurrencefor each biochemical and, when matched with an unknown, the computer canprovide a diagnosis based on probability. Minimum level of probabilitycan establish the acceptability of the answer provided. This approachrequires a huge memory which imposes the use of a computer and has thesetback of describing combinations of reactions which are mathematicallypossible, but never encountered in organisms.

The Profile Recognition Method of this invention also tests theorganisms simultaneously with a large number of biochemical characters,but only describes combinations which are most probably to occur withactual organisms. For example, for twenty biochemical reactions, thecomputer would allow 1,048,576 combinations. On the other handtraditional flow diagram techniques would only provide less than onehundred combinations. The Profile Recognition Method of the inventionprovides about 1500 combinations which is a more realistic number ofcombinations considering the probable number of biotypes.

The method of this invention enables the user to convert the results ofthe biochemical tests into a Profile Recognition Number, which isutilized to identify the bacterium by consulting a Profile Register.Methods are provided in the Profile Register to account for rare strainsof bacteria as well as to aid in correcting errors in interpreting thebiochemical test results made by the user.

The identification process can be further simplified by use of a coderto generate the Profile Recognition Number. This enhances the speed ofidentification.

Errors in transforming the biochemical test results into the ProfileRecognition Number may be made by the user. To reduce these errors, thecoder may also be provided with a color-check feature, whereby the usercan visually compare the colors produced in the reaction chambers with aset of colors generated on the coder corresponding to the indicatedProfile Recognition Number.

As used herein, the terms "numerical profile," "Profile RecognitionNumber," "number" and "digit" are understood to include both numericaland alphanumerical representations.

SUMMARY OF THE INVENTION

The Profile Recognition Method of this invention tests themicro-organism simultaneously with a large number of biochemicalcharacters, attributing to each of them the same weight. The ProfileRecognition Method not only considers each character, but also theprobability of simultaneous occurrence or mutual exclusion ofbiochemical characters. This method has long been advocated bytaxonomists.

This classification of micro-organisms such as bacteria, on the basis ofoverall similarities, has also long been advocated by microbiologists.Yet, it has not found a wide acceptance in the routine clinicallaboratory because of the work involved in performing a large number ofbiochemical reactions simultaneously and because of the complexity tointerpret the results gathered. The method of this invention allows thevery accurate performance of a large number of biochemical tests withconvenience, speed and economy.

The system allows standardization of the technique. The ProfileRecognition Method will now allow consistency of the interpretation.This method will increase further the confidence of the technologist inan accurate identification and a shorter reporting time to theclinicians for better patient therapy.

It is an object of this invention to provide a simple, fast, efficientand economical method for identification of bacteria. It is a furtherobjection to provide a more errorproof method for said identification.

It is still a further object to provide a more consistent method forrecognition of bacteria.

It is also an object to provide a simple and convenient coder formechanically aiding said identification.

It is still a further objection of this invention to provide a means tocheck on the accuracy of the identification.

Further objects will become apparent from the detailed specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a biochemical test strip of twentyindividual reaction chambers.

FIG. 2 is a perspective view of a representative section of the teststrip along the long axis of each chamber.

FIG. 3 is an enlarged vertical section view of an individual reactionchamber taken along the line 3--3 in FIG. 1.

FIG. 4 is a perspective view of a preferred form of the coder device.

FIG. 5 is a vertical section view of the coder device taken along theline 5--5 in FIG. 4.

FIG. 6 is an illustration of the seven indicator slides for thepreferred form of the coder device.

FIG. 7 is a representation of the steps of the method of the invention.

FIG. 8 is a perspective view of another coder device.

FIG. 9 is an illustration of one indicator slide for the coder device ofFIG. 8.

FIG. 10 is a representation of the time variation chart of thisinvention.

FIGS. 11A and 11B are representations of a time variation chart whichincludes the score values, the intermediate values and the final valuesof this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to a simple and efficient method for theidentification of micro-organisms including bacteria. The methodgenerally comprises the steps of culturing and isolating the unknownbacterium on a selective medium, reacting small portions of the culturewith preselected biochemical test reagents, transforming the results ofthese biochemical tests into a numerical profile, and then consulting atabulated list such as a catalog or register of numerical profiles(Profile Register) to identify the bacterium.

The unknown bacterium may be cultured by any convenient standard methodand in the usual nutrient broths. The culturing should be done for asufficient time to produce a reasonable concentration of bacteria in theculturing medium. The reliability of the biochemical tests will beincreased by allowing the medium to culture for a longer period of time.

After the desired culturing period, an individual colony is suspended inabout 2 to 5 milliliters of an appropriate diluent, which may or may notcontain a nutrient. This bacterial suspension is then transferred inmicro-quantities to the reaction chambers by any suitable means, such asa Pasteur pipette.

While the biochemical test reactions can be carried out in anyconvenient equipment and by any convenient method, it is preferably tocarry out the test reactions in a special plate manufactured for thepurpose. One such test plate is manufactured by Analytab Products, Inc.of Carle Place, Long Island, New York.

As shown in FIG. 1, the test plate 11 comprises a plurality of smallidentified reaction chambers 12 (identified by numbers for example)disposed linearly along the strip. As shown in FIG. 2, the strip isconveniently formed attaching two molded plastic strips 13 and 14 so asto form individual compartments 15. FIG. 3 illustrates the configurationof each reaction chamber.

As supplied by the manufacturer, there is a measured quantity of testreagent 16 and possible nutrients situated in the closed end of eachreaction chamber. It is most convenient if the reagent mixture thereinis in solid form (dehydrated), as then the test plates may be moreeasily stored and handled. As some of the reagent mixtures tend to beslightly unstable, it is preferable to keep the unused test platesrefrigerated until ready for use.

Referring again to FIG. 1, each test plate 11 contains a plurality ofmicro-reaction chambers 12. Each micro-reaction chamber along the platehas a different predetermined reaction mixture contained therein. Thuseach biochemical test can be performed simultaneously. The test platescan be supplied with differing numbers of tests. Rapid identificationhas been done with a series of ten tests and more accurate ones havebeen performed using a series of twenty tests and even fifty tests.

The sequence of twenty micro-tests for enterobacteriaceae has been about88% accurate, and reaches about 93% when using a more concentratedliquid culture. See, Washington, J. A. et al, Evaluation of Accuracy ofMultitest Micromethod System for Identification of Enterobacteriaceae,Applied Microbiology, 22:267-9 (Sept. 1971) A later investigatorobtained 96.4% accuracy. Smith, P. B. et al, API System: a MultitubeMicromethod for Identification of Enterobacteriaceae, AppliedMicrobiology, 24:449-52 (Sept. 1972)

TWENTY TEST METHOD

While any of these or other numbers of tests may be used with the methodof this invention, it has been found to be preferable to utilize aseries of twenty tests. The following description will illustrate theuse of one twenty-test method for the identification ofenterobacteriaceae, but it is important to realize that it is forpurposes of illustration only and may be adapted to any number of tests.

The twenty tests selected are:

1. B-galactosidase (ONPG)

2. arginine dihydrolase (ADH)

3. lysine decarboxylase (LDC)

4. ornithine decarboxylase (ODC)

5. citrate (CIT)

6. h₂ s

7. urease (URE)

8. tryptophane deaminase (TDA)

9. indole (IND)

10. acetoin (VP)

11. gelatin (GEL)

and fermentation tests of

12. glucose (GLU)

13. mannitol (MAN)

14. inositol (INO)

15. sorbitol (SOR)

16. rhamnose (RHA)

17. saccharose (SAC)

18. melibiose (MEL)

19. amygdaline (AMY)

20. arabinose (ARA)

Commercially available test strips containing the reagents and nutrientsnecessary to perform the above twenty biochemical tests are availablefrom Analytab Products, Inc., Carle Place Long Island, New York,referred to as the API Enteric System.

Also available from Analytab Products, Inc. are test strips containingmaterials to conduct only ten of the above biochemical tests. These tenbiochemical tests can provide quick test results with some sacrifice ofaccuracy compared to the twenty-test method.

For anaerobic bacteria, a similar set of twenty biochemical tests isavailable. The following list represents the specific biochemical testswhich are provided for in the available strips:

1. Indole

2. Urea

3. Glycerol

4. L (+) arabinose

5. D (+) xylose

6. Glucose

7. Mannose

8. Rhamnose

9. Mannitol

10. Sorbitol

11. Gelatine

12. Esculine

13. Salicine

14. Cellobiose

15. Maltose

16. Lactose

17. Saccharose

18. Trehalose

19. Melezitose

20. Raffinose

The above three sets of available test strips are meant to berepresentative only. Other sets of biochemical, chemical enzymatic,etc., tests can be utilized. The number of tests in the set is notcritical, but should be of sufficient number to produce reasonableaccuracy.

The order of tests on the test strip is of no consequence as all testsare performed simultaneously and are given equal weighting for the finalidentification. While the ordering of tests on the test strip isimmaterial, once an order is selected, it is important to consistentlyuse this same order. In each case, the Profile Register will depend onthe ordering of tests on the strip.

The description of the methods of this invention below will beillustrated by using the API Enteric System of Analytab Products, Inc.This is meant to be illustrative only and the methods of this inventionare equally applicable to any set of biochemical tests.

Referring again to FIG. 3, each reaction chamber 12 is filled with thebacterial suspension using the open end 17 of each reaction chamber 12.No attempt is made to agitate or otherwise distribute the contents. Theentire test plate 12 is then placed into an incubation chamber andallowed to incubate for about 18 to 24 hours at a temperature range ofabout 35° to 40°C. The incubation chamber can be close-fitting with thetest plate and may contain water, thereby reducing evaporation from thereaction chambers.

When the test strip for anaerobic bacteria is used, provision is made inthe incubation chamber (not shown) to replace the air contained thereinwith a suitable non-oxidizing atmosphere. Thus the test strip is placedinto the special incubation chamber, the chamber sealed, the airreplaced by a non-oxidizing gas and the test performed. In this case,the incubation chamber is preferably manufactured from a transparentmaterial in order to facilitate the checking of reaction results withoutintroducing air into the system.

For each test, the presence or absence of a reaction is detected. In thechart for enterobacteriaceae below, positive means that a reaction hastaken place in the numbered test chamber and negative means the absenceof any reaction.

By merely checking the color, except for test 11 where the presence orabsence of diffusion constitutes the test, the user can easily determinethose tests giving a positive result and those giving a negative result.

    ______________________________________                                        TEST             POSITIVE    NEGATIVE                                         ______________________________________                                         1.   ONPG (Lactose) Yellow      Clear                                         2.   Arginine       Red         Yellow                                        3.   Lysine         Red         Yellow                                        4.   Ornithine      Red         Yellow                                        5.   Citrate        Blue        Green                                         6.   H.sub.2 S      Black       Clear                                         7.   Urea           Red         Yellow                                        8.   Tryptophane    Brown       Yellow                                        9.   Indole         Red         Yellow                                       10.   Voges-Proskauer                                                                              Red         Clear-Pink                                   11.   Gelatin        diffusion   no diffusion                                 12.   Glucose        Yellow      Blue-Green                                   13.   Mannitol       Yellow      Blue-Green                                   14.   Inositol       Yellow      Blue-Green                                   15.   Sorbitol       Yellow      Blue-Green                                   16.   Rhamnose       Yellow      Blue-Green                                   17.   Saccharose     Yellow      Blue-Green                                   18.   Melibiose      Yellow      Blue-Green                                   19.   Amygdaline     Yellow      Blue-Green                                   20.   Arabinose      Yellow      Blue-Green                                   ______________________________________                                    

It is also possible to perform an oxidase (OXI) test as an additionaltest. This test can be performed in either chamber No. 1 (ONPG) or No. 6(H₂ S) whenever either is negative. As used herein, this test will bereferred to as test No. 21.

Referring to FIG. 7, which is an outline of the method of thisinvention, the test results are arranged into a plurality of groups. Ithas been found to be preferable to arrange the tests into groups ofthree. Thus the above twenty-one tests would produce seven groups ofthree tests each.

While the arranging by groups of three tests each is not essential, thenumber within a group should maximize both simplicity and efficiency.For example, should the tests be segrated into groups of four testseach, the resulting numerical profile will either be in hexadecimalnotation, which could be more difficult to the untrained or will entailmore than a single digit. Larger groupings could become absurd. Shouldsmaller groupings be selected, then more digits will be required than bythe grouping by threes. The grouping by three tests each of the methodof this invention enables the transforming of the test results of thegroup into a single score such as a single digit. Each test within agroup can be assigned a weighted score. The selection should be made sothat the sum of any combination of scores is unique and preferably of asingle digit. For example, assigning a score of zero to all negativeresults and score of 1, 3 and 5 to positive results of each of the threetests, respectively, will produce a unique set of such sums.

While any such set of scores will suffice, it is preferred to assign thefollowing scores. These twenty-one tests of the API Enteric System plusthe oxidase test are divided into seven groups of three:

    ONPG  ODC      URE     VP     MAN   RHA    AMY                                ADH   CIT      TDA     GEL    INO   SAC    ARA                                LDC   H.sub.2 S                                                                              IND     GLU    SOR   MEL    OXI                            

After the results have been reported, a score is given to each positiveresult:

A score of ONE for the first biochemical of any group of three (i.e.,ONPG, ODC, URE . . . )

A score of TWO for the second biochemical of any group of three (i.e.,ADH, CIT, TDA . . . )

A score of FOUR for the third biochemical of any group of three (i.e.,LDC, H₂ S, IND . . . )

All negative tests score zero, producing the following for eachcombination of results:

    Test Results         Sum                                                      ______________________________________                                        All negative         0                                                        No. 1 positive, No. 2, 3 negative                                                                  1                                                        No. 2 positive, No. 1, 3 negative                                                                  2                                                        No. 1, 2 positive, No. 3 negative                                                                  3                                                        No. 3 positive, No. 1, 2 negative                                                                  4                                                        No. 1, 3 positive, No. 2 negative                                                                  5                                                        No. 2, 3 positive, No. 1 negative                                                                  6                                                        All positive         7                                                        ______________________________________                                         Each digit of the seven-digit number is obtained by totaling the score of     the positive reaction of each group of three. Example: 5 044 552 = E. coli     ##EQU1##     It should be noted that each number corresponds to only one combination     and, therefore, for each seven-digit number there will be only one     corresponding profile.

In effect, this selection of scores results in the conversion of thebinary results (positive or negative) of each test into a single octaldigit. Thus, the grouping of these twenty-one tests in the API EntericSystem into groups of three will produce seven individual octal digitswhich will characterize all twenty-one tests. Thus the usercharacterizes each test as positive or negative and assigns the scorefor each test in the group. Then the scores for each in the group areadded together, thereby producing a one-digit number. For example, if ingroup 4, test 10 is positive, 11 is negative and 12 is positive, thentest 10 has a score value of one, test 11 scores zero, and test 12scores four. Adding the scores together, the score of five is obtainedfor test grouping number 4.

The digits representing the sum of the scored for each test group arecombined to form a seven-digit octal number. The seven digits arewritten down sequentially. Thus, the digit from test group 1 becomes thehigh order digit, the digit from test group 2 is next, etc., and thedigit from test group 7 becomes the lowest order digit. Naturally anysimilar predetermined method of combining these digits will suffice. Theseven-digit number thus produced is the Profile Recognition Number.

A Profile Register 18 (FIG. 7) of sequentially listed ProfileRecognition Numbers is then consulted and the bacterium identified. Asan example, if tests 1, 9, 12, 13, 18 and 20 are positive and theremainder of tests negatives, the identification proceeds as follows:

Group No. 1 has its first test positive and second and third negativeand gets a score of 1.

Group No. 2 is all negative and receives a score of 0.

Group 3 has its third test positive and others negative and receives ascore of 4.

Group No. 4 also has its third test positive and others negative andreceives a score of 4.

Group No. 5 has its first test positive only and receives a score of 1.

Group No. 6 has its third test positive only and receives a score of 4.

Group No. 7 has its second test positive, others negative and receives ascore of 2.

Combining all digits produces the Profile Recognition Number 1044142.The Profile Register identifies this number as being associated with thebacterium E. coli.

A profile Register of pre-identified profiles has been established. Theestablishment of these pre-identified profiles has first been performedon a theoretical basis and then matched with more than 25,000 resultsobtained using the API 20 Enteric device.

Initially a theoretical register has been established based onpercentage data which has been previously reported in technicalpublications. Reactions have been considered as variable whenever apercentage of positive reactions was 95% or less. Profile numbers havebeen established considering all possible combinations obtained bypermutation of the variable reactions. If, for example, three reactionswere variable, eight profile numbers have been established. For eightvariable reactions, 256 profile numbers have been established. Thepurpose of this theoretical register was to determine the possibilityfor one profile number to correspond to more than one organism. Becausetwenty biochemicals are considered simultaneously, we encountered veryfew such occurrences.

Results obtained on this system, mainly from clinical laboratories, havebeen gathered. These results came mainly from the following countries:the United States, Canada, France, England and Germany. Inasmuch aspossible, results have been gathered from several geographical areas.For example, results from the U.S. were obtained from the followingstates: California, Georgia, Louisiana, Maine, Michigan, Minnesota,Nebraska, New Jersey, Texas and Washington, D.C.

Whenever more than one organism name has been reported for the sameprofile number, consideration of theoretical results and probabilityhave been considered and whenever necessary and possible, the organismhas been run through the API 50 Research system which provides for aprofile based on fifty biochemicals.

Whenever organisms could not be separated on the basis of the 20biochemicals, reference is being made to a note which suggestscomplementary tests required to separate such organisms.

The Profile Register is used as follows:

After a profile number has been recorded, either using the mathematicalconversion method or the Coder device 22 (FIG. 4), discussed below, thenumber is then searched in the Profile Register.

1. Whenever a number of an unknown is matched with a number of theProfile Register, there is an extremely high probability that thedesignation of this organism is the one indicated by the ProfileRegister. The three last digits of the profile number may appear ineither small or larger typeface, or in large typeface underlined. Theunderlined large typeface indicates a very common biotype. The largetype (without underlining) indicates a common biotype and the smalltypeface indicates a rare biotype.

Example: 5144552- E. coli -very common biotype 5144550 - E. coli -commonbiotype 5 004 552 - E. coli -rare biotype (IND -)

2. Whenever a number refers to another number, it is implied that abiochemical test has been misinterpreted. The user should refer to thecorrect number for proper identification and go back to themisinterpreted results for further improvement of color interpretation.

Example: 7 304 733 - See 5 304 733

The difference is only the first digit ##EQU2## This indicates that thearginine of orange color should have been interpreted as negative for S.liquefaciens.

3. If the number obtained is not in the Register, the number shouldfirst be rechecked using both the mathematical and Coder approaches. Ifthe number is still not in the Register, it will mean that the strain isa very rare biotype which has not yet been listed in the Register orthat there is an error in the laboratory procedure. In this case, theapproach of a diagnosis by elimination should be considered and, if notconclusive, a new test strip should be rerun after a new isolation hasbeen made and the results compared. If the number then obtained is thesame as initially which does not fit any preset pattern, this organismshould be sent to competent authorities for further identification. Atthis time, the use of the API 50 Research system can prove to be veryuseful.

When an organism does not fit the profile expected, diagnosis byelimination can be considered. This approach is to try to match theunknown profile with each of the species of the familtyEnterobacteriaceae and see the reason why this unknown profile cannot besuccessively each of them.

The selection system, as discussed below, has been based on theexperience gathered in studying more than 25,000 organisms for theestablishment of the Profile Register. The numbers indicated in blackfor each organism are numbers which correspond to very common biotypes.The black numbers in any column represent about 90% of the organismsencountered for this specie. The numbers indicated in blue are numbersrepresenting very seldom encountered biotypes. These blue numbersrepresent percentages of occurrence generally between 1 and 5%.

EXAMPLE:

0 054 210 -- This profile is not found in the Profile Register. The useof the Selector will permit the following deduction and elimination:

a. because of its two first digits, "0 0", this unknown should not be S.sonnei, Salmonella, P. mirabilis, P. morganii, KES group, Citrobacterspecies. Actually, what we are saying is that the above-mentionedorganisms must have at least one positive reaction within the first sixbiochemical tests on the API 20 Enteric system. By elimination, it canonly be: E. coli, Shigella, P. vulgaris, P. rettgeri, Providencia,Pectobacterium, Y. enterocolitica.

b. because of its third digit, "5", this unknown should not be E. coli,Shigella, Providencia. By elimination, it can only be: P. vulgaris, P.rettgeri, Pectobacterium or Y. enterocolitica.

c. the fourth digit, "4", is of no assistance to eliminate any of thesefour organisms.

d. the last three digits, "210", eliminates Pectobacterium and Y.enterocolitica. This unknown can therefore theoretically be either a P.vulgaris or a P. rettgeri. We can now consider what the highestprobability for each of these organisms is by counting the black andblue figures.

    ______________________________________                                        P. vulgaris   3 black       4 blue                                            P. rettgeri   5 black       2 blue                                            ______________________________________                                    

The highest probability is that this organism is a P. rettgeri which canbe verified by checking the result with the API Per Cent Chart.

Referring now to FIG. 4, the apparatus represented therein is thepreferred form of a coder 22 to partially automate the above method. Thecoder allows the conversion of positive and negative results into theProfile Recognition Number.

Preferred embodiments of the Coder as shown in FIGS. 4, 5 and 8 comprisea base member 22a fixedly attached on three sides to a cover member 22b,in such manner as to form a support frame and to leave a thin flatelongated chamber 22c defined by these members. The material may be anyconvenient one such as acrylic (Plexiglass) material, but should befairly rigid and is preferably opaque. A plurality of rib members 22dare fixedly attached within the defined chamber to provide for aplurality of equal sized chambers. It is most convenient for carryingout the above method if the rib, base and cover members define sevenindividual chambers as shown in FIG. 4. The embodiment shown in FIG. 8where five individual chambers are used is the preferred embodiment ofthe Coder when using the fifty test method, described below.

A slidably mounted flat member 23 or slide is fitted into each of thechambers 22c. Thus if seven chambers are provided, seven slide members23 are provided. The slide members can be of such size as to fit closelyto the dimensions of the chambers as to be held therein by friction, forexample, should the entire apparatus be disposed with the open slidedownward. Conversely each slide member can be easily moveable by meansof a pencil, probe, stylus or the like 24, inserted in indentations 23ain each slide member (FIG. 6). The number of chambers and number ofslide members may differ from seven when using other than the APIEnteric System. The seven slide members of the apparatus of thisinvention correspond to the seven test groups described in the abovemethod. Thus, while each slide member is structurally identical to eachother slide member, each may differ in marking in accordance with thespecific biochemical test represented thereon.

FIG. 6 is a representation of the seven slide members corresponding tothe 21 tests of the API Enteric System described above. Each slidemember 23 is generally of elongated rectangular top surface with athickness adopted to fit its related chambers.

The slides 23 are fitted with a plurality of indentations 23a in orderthat the slide may be moved by inserting the tip of the stylus andpushing the slide. These are disposed into three columns of equallyspaced indentations. The three columns represent the three tests of thegroup. Should some other grouping be used, suitable modification must bemade in the slide member. Each slide member is also imprinted thereonwith the possible digits 23b representing the sum of the scores for eachtest. When used with the illustrative tests described herein, the digitswill be 0 through 7. The digits are placed on the slide member in suchorder that the correct digit will be generated whenever the slide memberis moved in accordance with the instructions below. For the API EntericSystem, they are placed sequentially, reading from the bottom of themember as shown in FIG. 6.

The cover member 22b shown in FIG. 4 contains a series of three slots25, 26, 27 corresponding to each slide 23. There is also a window 28overlying each slide member. The slots are in appropriate lengths orincrements to linearly represent the scores designated for each of thethree tests of the group. With the system herein described, the slots25, 26, 27 generally have lengths or increments in the ratio of 1:2:4respectively. The slots 25, 26, 27 and the window 28 are disposed insuch manner that when the slide member is mounted in the Code 22,apparatus the indentations in the slide member are accessible throughthe slots and one digit imprinted on the slide member is visible throughthe window.

The cover member 22b may also contain 21 additional windows 29 disposedin such manner that three correspond to each slide member. The number ofsuch windows will be the same as the number of tests being performed.Each slide member is provided with three columns 30, 31 and 32 of eightareas 30a-h, 31a-h and 32a-h, respectively, each for color coding. Whenthe slide member is inserted into the apparatus, one color area of eachcolumn on the slide member will be visible through the correspondingwindow 29 in the cover member.

The colored areas can be determined as follows:

Color column 30 relates to the first test of the test group and colorareas 30a, 30c, 30e and 30g (from bottom) of the column are colored thenegative color of this test, while areas 30b, 30a, 30f and 30h receivethe positive reaction color.

Column 31 relates to the second test of the group. Areas 31a, 31b, 31eand 31f are imprinted with the negative color and areas 31c, 31d, and31g and 31h with the positive.

Column 32 relates to the third test of the group. Areas 32a, 32b, 32cand 32d are in the negative color and areas 32e, 32f, 32g and 32h are inthe positive one.

Each biochemical reaction corresponds to a slot (25, 26, or 27) andwindow 29 on the cover 22b (FIG. 4). Each group of three reactionscorresponds a slide 23 and a window 28. The operating principle of thecoder 22 is to move the slide parts only when the reactions arepositive. The apparatus is made ready for use by having all slidemembers fully inserted into the base-cover combination. At this time,the digits 23b (FIG. 6) visible in each of the windows 28 (FIG. 4) mustbe zero. Likewise, the colors visible in windows 29 must all be thecolors indicating the negative for each reaction. The cover member 22bidentifies each elongated hole 25, 26 and 27 and each window 29 with thetest to which it corresponds. These are arranged in numerical order fromleft to right or viewed in FIG. 4.

The reactions are reported from the left to the right. When a reactionis positive, the pen is inserted in the upper part of the slot andlowered to the extremity of the slot, moving the slide therewith. If areaction is negative, the sliding part remains unmoved. After eachreaction has been reported, a color matching the positive reactionobtained on the API strip should appear in the corresponding opening.

After each group of three biochemicals have been reported, the openingon the lower part corresponding to the three biochemicals will show theproper number which will be one of the seven digits number. Uponcompletion of recording of the 21 biochemical reactions, the Coder 22will provide the user with a seven digit number visible in the sevenwindows 18.

The Coder has a built-in verification system which allows the matchingof color patterns obtained on the test strip 11 with the color patternobtained on the Coder 22 after the slide members have been moved inaccordance with instruction. The colors are visible in windows 29.

In case a reaction has been mis-recorded, it is suggested that the threereactions corresponding to the sliding part where the mistake occured beplaced back into original position and the three reactions recordedproperly. For example: If the reaction ADH is mis-reported, it isrecommended that the first slide member corresponding to the reactionONPG, ADH, LDC be placed in its original position.

After completion of the reporting, the API Coder should be returned toits initial position by placing the Coder vertically and pushing it downin order to reinsert the sliding members.

FIFTY TEST METHOD

As hereinbefore stated, the method of this invention may be used tointerpret the results from any number of tests. It has been found that aparticularly sensitive test result may be obtained when using a seriesof fifty tests.

The following purely illustrative description will illustrate the use ofone fifty-test method for the identification of Enterobacteriaceae.

Test strips, essentially similar to that illustrated in FIG. 8, areavailable commercially from Analytab Products, Inc., Carle Place, NewYork 11514 and sold under the name API 50 Research System.

The following description will illustrate the method of the inventionusing the API 50 Research System. It is understood, however, that anysimilar set of tests may be utilized.

It is not necessary that the user utilize a commercially available teststrip. It is within the intent and scope of this invention to describe amethod of conversion of test results into a numerical profile. It isentirely possible to utilize no prepared test strip, but to perform theindividual tests separately in culture tubes or the like. This latterprocedure will however, reduce the speed and convenience of the overallmethod.

The ordering of the tests on the test strip is of no consequence as alltests are performed simultaneously and are given equal weight inevaluating the results. However, it is necessary to arbitrarilypreselect a given sequence and to adhere to that sequence. The ProfileRegister tabulations will depend on the order selected.

The API 50 Research System utilizes the following fifty tests in theorder listed.

1. phenol red-control tube

2. glycerol

3. erythoritol

4. d(-) arabinose

5. L (=) arabinose

6. ribose

7. d (+) xylose

8. L (-) xylose

9. adonitol

10. methyl-xyloside

11. galactose

12. d (+) glucose

13. d (+) levulose (fructose)

14. d (+) mannose

15. L (-) sorbose

16. rhamnose

17. dulcitol

18. meso-inositol

19. mannitol

20. sorbitol

21. methyl-d-mannoside

22. methyl-d-glucoside

23. N acetyl-glucosamide

24. amygdalin

25. arbutine

26. esculine

27. salicin

28. d (+) cellobiose

29. maltose

30. lactose

31. d (+) melibiose

32. saccharose (sucrose)

33. d (-) trehalose

34. inuline

35. d (+) melizitose

36. d (+) raffinose

37. dextrine

38. amylose

39. starch

40. glycogen

41. ONPG

42. arginine

43. lysine

44. ornithine

45. citrate

46. thiosulfate

47. urea

48. tryptophane

49. tryptophane-peptone

50. pyruvic acid

Reaction chambers Nos. 1-25, 25-40, 42-44 and 47 also contain phenol redas an indicator. The material in reaction chamber No. 25 includes amixture of phenol red and ferric chloride and chamber No. 26 containsferric chloride as indicators. Reaction chambers Nos. 41, 45-46 and48-50 do not contain an indicator.

The above-described test strips are particularly designed for thedetermination of enterobacteriaceae. The first forty tests arecarbohydrate fermentations. The same test strips may also be used forthe identification of bacterial families for which carbohydratemetabolism is important, such as streptococcus, staphylococcus,pasteurelleae, vibrionaceae and some pseudomanadaceae.

Tests 41 through 50 are identical to tests 1-10 of the previouslydescribed API Enteric System.

The test strips for the API Research System include nutrients inreaction chambers Nos. 41-50. No nutrient is included in reactionchambers Nos. 1-40, thereby providing a greater versatility to the teststrips. The user can then utilize differing nutrient media depending onthe particular bacterium being tested.

The bacterium may be cultured by any convenient method. If it iscultured on an agar plate, small sections may be selected with a wireloop for suspension. If cultured in a liquid medium, then it ispreferable to centrifuge the liquid to obtain a more concentratedculture. In any event, it is necessary to use proliferant bacteria in anexponential phase growths.

The sample of culture bacteria is then suspended into a suitable medium.It is necessary to prepare two different suspension media for use withthis test strip.

The first suspension is prepared for use in research chambers Nos. 1-40.These chambers do not contain any culture medium. Hence the bacterialsuspension for use with these chambers must include the culture medium.In this manner, the system has a greater flexibility of use withdifferent types of bacteria, by allowing the use of differing culturemedia depending on the suspected identity of the unknown bacterium.

The culturing medium selected must be rich enough to promote the rapidgrowth of the bacteria. It should not contain any fermentativesubstances and preferably has a pH of about 7.4. Classical culturingmedia may be used. For example, when the bacterium is a suspectedenterobacteriaceae a standard peptane broth may be used. It ispreferable to use a peptane broth enriched with yeast extract when theunknown bacterium is suspected to be one which is more difficult togrow, such as a streptococcus.

The bacterial culture suspension should be sufficiently dense to permitmultiplication, but not excessively dense. Generally, its opticaldensity should be about the same as tube 1 of the standard McFarlandoptical density scale.

The McFarland scale is prepared by introducing different quantities of1% barium chloride and 1% sulfuric acid into ten identical tubes. Forexample, ten 16mm test tubes are used. It is convenient if they aregraduated to contain 10ml. 0.1 ml. of the barium chloride solution isintroduced into the first tube, 0.2ml. into the second and so on up to1.0ml. added to the tenth tube. Each tube is then filled to the 10mlgraduation with the sulfuric acid solution, the tube stoppered and thecontents thoroughly mixed. It will be necessary to shake the tubes priorto each use to distribute the sediment throughout the liquid. Thebacterial suspension and the McFarland standard tubes are then visuallycompared.

If both oxidation and fermentation of the bacterium in the presence ofthe various carbohydrates is studyed, then the suspension medium ispreferably 0.7% agar. The suspension is prepared by warming the mediumto about 40°C and the bacterial culture introduced. The suspension mustthen be inoculated into the reaction chambers while still likewarm,before gelification.

Reaction chambers Nos. 41-50 contain, in addition to the aforementionedmaterials, the culture medium. Hence none is to be added to thebacterial suspension designed for use in these chambers. It ispreferable that the bacteria be suspended in either distilled water ornormal saline solution. A suspension having an optical density of tube 1on the McFarland scale is preferable.

The reaction chambers are inoculated by introducing the appropriatebacterial suspension into the interior of each reaction chamber, such asby use of a Pasteur pipette. Care should be taken to avoid theintroduction of air bubbles into the suspension.

It is preferable that each reaction chamber be filled properly. Thequantity of suspension to be introduced will vary with the suspectedidentity of the unknown bacterium.

Referring again to FIG. 3, each reaction chamber 15 can be visualized asbeing divided into regions: the tube section 15a, and the cupule 15b,which is that region encircled by flange 17.

Reaction chambers Nos. 1-40 are preferably filled with the bacterialsuspension in the following manner. It is generally only necessary tofill the tube region with the suspension. However, if the suspectedidentity of the bacterial family is one which is known to produce eithervolatile acids or relatively small quantities of acids, such asStreptococcus and Pasturella, the chamber should be sealed by fillingthe cupule with sterile paraffin oil. When the unknown bacterium issuspected to be one which both grows and produced acid, then both thetube region and the cupule are filled with the suspension. It is withsuch bacteria that the preferred culture medium is the ag 2.

Reaction chambers Nos. 41-50 are filled as follows regardless of thesuspected identity of the bacterium.

In reaction chambers Nos. 41-44 and 46-49, only the tube region isfilled. Both the tube region and the cupule are filled in chambers Nos.45 and 50. Reaction chambers Nos. 42-44 and 47 should be sealed byfilling the cupule with sterile paraffin oil.

The inoculated test plate is then incubated in the same manner asdescribed for the API Enteric System. It is preferable to incubate thetest plate for at least 18 hours before making a determination of theresults.

After the proper incubation period a determination is made of whetherthe bacterium has or has not reacted with each test reagent.

Prior to making this determination, several drops of Ferric chloride areadded to chamber No. 48; several drops of Kovaks reagent to chamber No.49 and several drops of a potassium hydroxide- and α-napthol solution tochamber No. 50.

The determinations of evidence of reaction are made by visuallyobserving the color of the material in a manner similar to thatdescribed for the API Enteric System.

    ______________________________________                                        Test                Positive   Negative                                       ______________________________________                                         1.   Phenol red          remains red                                          2.   Glycerol          yellow     red                                         3.   Erythoritol       yellow     red                                         4.   d(-) Arabinose    yellow     red                                         5.   L(+) Arabinose    yellow     red                                         6.   Ribose            yellow     red                                         7.   d(+) Xylose       yellow     red                                         8.   L(-) Xylose       yellow     red                                         9.   Adonitol          yellow     red                                        10.   Methyl-xyloside   yellow     red                                        11.   Galactose         yellow     red                                        12.   d(+) Glucose      yellow     red                                        13.   d(+) Levulose (Fructose)                                                                        yellow     red                                        14.   d(+) Mannose      yellow     red                                        15.   L(-) Sorbose      yellow     red                                        16.   Rhamnose          yellow     red                                        17.   Dulcitol          yellow     red                                        18.   Meso-inositol     yellow     red                                        19.   Mannitol          yellow     red                                        20.   Sorbitol          yellow     red                                        21.   Methyl-d-mannoside                                                                              yellow     red                                        22.   Methyl-d-glucoside                                                                              yellow     red                                        23.   N-acetyl-glucosamide                                                                            yellow     red                                        24.   Amygdalin         yellow     red                                        25.   Arbutine          yellow-black                                                                             red                                        26.   Esculin           black      colorless                                  27.   Salicin           yellow     red                                        28.   d(+) cellobiose   yellow     red                                        29.   Maltose           yellow     red                                        30.   Lactose           yellow     red                                        31.   d(+) Melibiose    yellow     red                                        32.   Saccharose (sucrose)                                                                            yellow     red                                        33.   d(-) Trehalose    yellow     red                                        34.   Inuline           yellow     red                                        35.   d(+) Melizitose   yellow     red                                        36.   d(+) Raffinose    yellow     red                                        37.   Dextrine          yellow     red                                        38.   Amylose           yellow     red                                        39.   Starch            yellow     red                                        40.   Glycogen          yellow     red                                        41.   ONPG              yellow     colorless                                  42.   Arginine          red        red                                        43.   Lysine            red        yellow                                     44.   Ornithine         red        yellow                                     45.   Citrate           blue       green                                      46.   Thiosulfate       black      colorless                                  47.   Urea              red        yellow                                     48.   Tryptophane       brown      yellow                                     49.   Tryptophane-peptane                                                                             red rind   yellow red                                 50.   Pyruvic acid      bright red colorless                                  ______________________________________                                    

Each determination that a reaction has assigned is assigned a value of +and each determination is assigned a value of -.

It is most convenient to group the fifty tests in groups of five. Thus,ten such groups are assigned. For example, the first group would includethe following five determinations.

1. Phenol red

2. Glycerol

3. Erythritol

4. d(-) Arabinose

5. L(+) Arabinose

For each group, there are 32 possible combinations of determinations ofwhether the specimen has or has not reacted. A set of 32 digits orintermediate values has been selected to represent each of the 32conditions within each group. Each digit thus represents the results ofone group of five tests.

The following list of digits has been selected. To avoid confusion, theletters i and o, and the numbers 1 and 0 have not been used. Of course,any list of representations may be used, but once selected, the samelist must always be used.

                  LISTING OF 32 SYMBOLS                                           ______________________________________                                        A       -        -        -      -      -                                     B       +        -        -      -      -                                     C       -        +        -      -      -                                     D       -        -        +      -      -                                     E       -        -        -      +      -                                     F       -        -        -      -      +                                     G       +        +        -      -      -                                     H       -        +        +      -      -                                     J       -        -        +      +      -                                     K       -        -        -      +      +                                     L       +        -        +      -      -                                     M       -        +        -      +      -                                     N       -        -        +      -      +                                     P       +        -        -      +      -                                     Q       -        +        -      -      +                                     R       +        -        -      -      +                                     S       -        +        +      +      -                                     T       +        -        +      +      -                                     U       -        +        +      -      +                                     V       +        +        -      +      -                                     W       +        -        +      -      +                                     X       -        +        -      +      +                                     Y       +        +        +      -      -                                     Z       +        +        -      -      +                                     2       +        -        -      +      +                                     3       -        -        +      +      +                                     4       +        +        +      +      -                                     5       +        +        +      -      +                                     6       +        +        -      +      +                                     7       +        -        +      +      +                                     8       -        +        +      +      +                                     9       +        +        +      +      +                                     ______________________________________                                    

Thus, for example, if in the first group there is a positive reactionwith Erythritol and none with the other reagents, the representation forthe group would be:

- - + - -

which yields the value of "D" from the listing.

A similar determination is then made for each of the ten groups ofdeterminations. The ten digits thus selected are combined to form a tendigit numerical representation which is indicative of the identity ofthe unknown bacterium. A preferred method of generating this numericalrepresentation or final value is to list the intermediate values inidentical order to that in which they are generated, starting with thelowest order test numbers.

For example, if after incubation, the following tests yield a positiveresult:

2. Glycerol

6. Ribose

11. Galactose

12. d(+) Glucose

13. d(+) Levulose

14. d(+) Mannose

16. Rahmnose

18. Meso-inositol

19. Mannitol

20. Sorbitol

23. N acetyl-glucosamide

24. Amygdalin

25. Arbutine

26. Esculin

27. Salicin

28. d(+) Cellobiose

29. Maltose

30. Lactose

32. Saccharose

33. d(-) Trehalose

35. d(+) Melizitose

41. ONPG

42. arginine

and the remainder negative, the result would be:

    Group   Results                 Value                                         ______________________________________                                        1       -       +       -     -     -     C                                   2       +       -       -     -     -     B                                   3       +       +       +     +     -     4                                   4       +       -       +     +     +     7                                   5       -       -       +     +     +     3                                   6       +       +       +     +     +     9                                   7       -       +       +     -     +     U                                   8       -       -       -     -     -     A                                   9       +       +       -     -     -     G                                   10      -       -       -     -     -     A                                   ______________________________________                                    

and the final value would be:

CB473 9UAGA

A Profile Register has been established for the API 50 Research Systemin similar manner to that established for the API Enteric System.Consulting the Profile Register for this system,

    CB473 9UAGA = Streptococcus

It is important to realize that the results of the determination maydepend upon the nature of the culture medium in which the bacterium issuspended. It is thus a further feature of the Profile Register toenable the user to obtain the correct identification of regardless ofthe culture medium used.

It has been found convenient to identify the correct tabular listing bymeans of color coding the printing in the Profile Register. For example,the following printing colors can be utilized.

    Suspension Medium  Print Color                                                ______________________________________                                        tryptocose broth   Black                                                      yeast extract broth                                                                              Blue                                                       nitrated broth     Green                                                      ______________________________________                                    

While the above color-coding method has proven convenient, the sameresult can be obtained by other means, such as by using standard type,italics, bold face, etc.

The step of converting the score values for each group of determinationsinto the intermediate value can also be performed in a manner similar tothe mathematical method described for the example of the API EntericSystem.

When using such method with the API 50 Research System, thedeterminations are grouped in groups of five test each. Each of the fivetests within the group is then assigned a weighted score value.

It is convenient to assign score values to each determination where thefirst determination within a group receives a score value of 1, thesecond a score value of 2, the third 31 4, the fourth -8 and the fifth-16. The intermediate value can then be the sum of the score valueswithin the group. This method is inconvenient as the intermediate valuefor each group may not be a single digit.

However, this method of forming the intermediate value is utilized whenusing a mechanical coder device similar to that used for the API EntericSystem.

A coder device useful with the 50 test system could be constructedutilizing ten chambers and slide members, each corresponding to a singletest group. However, such coder would be cumbersome and unwiedly. It isthus preferable to utilize a coder having only five slide members,wherein score values of the first five groups of determinations areconverted to the five intermediate values, the slide members replacedand the process repeated for the second set of five groups.

Referring to FIG. 8, the preferred form of the coder used with the 50test system is generally constructed similarly to the coder (FIG. 4)used with the 21 test system.

Five chambers 22c are provided into each of which a single slidablymounted flat member 23 is fitted.

FIG. 9 is a representation of a single slide member 23 adapted for usewith the coder of FIG. 8. Each slide member is designed to fit snuglyinto its respective chamber in the coder.

Each slide member is fitted with a plurality of indentations (not shown)in order that the slide may be moved by inserting the top of a stylusand moving the slide. These are disposed in five columns of equallyspaced indentations. The five columns represent the five tests of thegroup.

Each slide member is also imprinted with a set of indicia 41,representing the 32 possible combinations of test results within thegroup. The indicia are ordered as shown to be consistent with the methodpreviously described for converting the score value to the intermediatevalue and to allow the use of the same Profile Register.

The cover member 22b (FIG. 8) contains a series of five slots 42, 43,44, 45, 46 corresponding to each slide. There is also a window 47overlying each slide member. The slots are of such lengths as tolinearly represent the weighted score value for each test within thegroup. Generally, for the described system, they will be in the ratio of1:2:4:8:16. The slots are disposed in such manner as to allow the tip ofa stylus to be inserted through the slot into the indentations in theslide member. The window 47 is disposed in such position so that one ofthe indicia 41 is visible through the windows.

The cover member 22b may also contain five additional windows 48 foreach chamber 22c. Each slide member has imprinted thereon five columns49 of 32 characters each. When the slide member is inserted into eachchamber, one character from each column is visible through each window48.

The coder device of the 50 test system is operated in a manner similarto that described for the 20 test system. The intermediate value foreach group of tests is read through windows 47.

The characters 49 visible through windows 48 provide a check for theoperator. The characters visible through these window will correspond towhether the unknown bacterium has or has not reached. A - visiblethrough the window can indicate an absence of reaction and a + thepresence of reaction.

These areas on the slide can of course be color coded with the colorsindicating whether the bacterium has or has not reacted, as describedpreviously.

A generally similar test plate is also commercially available fromAnalytab Products, Inc., Carle Place, New York, which is adopted for usewith Lactobacillus. In this system, the following tests are utilized,and the corresponding colors.

    ______________________________________                                        Test              Positive   Negative                                         ______________________________________                                         1. Bromcresol purple   remains purple                                         2. Glycerol          yellow     purple                                        3. Erythoritol       yellow     purple                                        4. d(-) Arabinose    yellow     purple                                        5. L(+) Arabinose    yellow     purple                                        6. Ribose            yellow     purple                                        7. d(+) Xylose       yellow     purple                                        8. L(-) Xylose       yellow     purple                                        9. Adonitol          yellow     purple                                       10. Methyl-xyloside   yellow     purple                                       11. Galactose         yellow     purple                                       12. d(+) Glucose      yellow     purple                                       13. d(+) Levulose (Fructose)                                                                        yellow     purple                                       14. d(+) Mannose      yellow     purple                                       15. L(-) Sorbose      yellow     purple                                       16. Rhamnose          yellow     purple                                       17. Dulcitol          yellow     purple                                       18. Meso-inositol     yellow     purple                                       19. Mannitol          yellow     purple                                       20. Sorbitol          yellow     purple                                       21. Methyl-d-mannoside                                                                              yellow     purple                                       22. Methyl-d-glucoside                                                                              yellow     purple                                       23. N-acetyl-glucosamide                                                                            yellow     purple                                       24. Amygdalin         yellow     purple                                       25. Arbutine          yellow black                                                                             purple                                       26. Esculin           black      colorless                                    27. Salicin           yellow     purple                                       28. d(+) cellobiose   yellow     purple                                       29. Maltose           yellow     purple                                       30. Lactose           yellow     purple                                       31. d(+) Mellibiose   yellow     purple                                       32. Saccharose (sucrose)                                                                            yellow     purple                                       33. d(-) Trehalose    yellow     purple                                       34. Inuline           yellow     purple                                       35. d(+) Melizitose   yellow     purple                                       36. d(+) Raffinose    yellow     purple                                       37. Dextrine          yellow     purple                                       38. Amylose           yellow     purple                                       39. Starch            yellow     purple                                       40. Glycogen          yellow     purple                                       41. Arginine          red        yellow                                       42. Glucose           bubbles    no bubbles                                   43. Teepol 0.4%       growth-yellow                                                                            inhibition-purple                            44. Teepol 0.6%       growth-yellow                                                                            inhibition-purple                            45. Na Cl 4%          growth-yellow                                                                            inhibition-purple                            46. Na Cl 6%          growth-yellow                                                                            inhibition-purple                            47. Na Cl 10%         growth-yellow                                                                            inhibition-purple                            48. ONPG              yellow     purple                                       49. KNO.sub.3 + glucose                                                                             red        colorless                                    50. Pyruvic Acid      red        colorless                                    ______________________________________                                    

The color indicator for audification used in this system is bromcresolpurple, which is included in reaction chambers Nos. 1-25, 27-40 and42-47. Phenol red is used in chamber No. 41 and iron citrate is presentin chambers Nos. 25 and 26.

This system is particularly designated to enable the user to determinethe particular strain of the lactobacillus.

The invention includes a method for the identification of lactobacilliby cultural and biochemical characteristics. It can also be used for theidentification of lactic streptococci and leuconostoc (betacocci).

To obtain reproduceable results, the experimental conditions must remainidentical. These are generally easy to obtain. All the strains are grownon the same medium, during the same time and at the same temperatures.The in-growth culture is washed by centrifugation. With the sediment, abacterial suspension is prepared in the "identification" medium definedby its optical density or numeration. The suspension is inoculated ineach of the microtubes of identical capacity. During incubation, thespeed of the appearances of the biochemcial reactions is reported on aresult sheet forming a profile (FIG. 10). A comparison of the obtainedprofile with the reference profile enables control of the production.

It is necessary to employ a satisfactory nutritional medium. Theidentification can only be performed on strains which are in continuousgrowth for several days and in the phase of exponential growth.

The suspensions prepared should contain an identical number of bacteria.The optic density can be measured with a photometer (at 525 nm theappropriate density is about 0.200 in a 1.0 cm cell). For greaterprecision, particularly in the suspension of the industrial productionof the same strain, the bacteria are counted by using a Coulter counter.For practical usage, the density can be compared to the McFarland scale.

The plate 11 is sterile. Each plate contains 50 biochemical reactions.The plate is then placed into a plastic box provided with the plate andsome water is poured into the bottom of the box to create a humidatmosphere.

Using a Pasteur pipette, all the tubes are filled avoiding theintroduction of air bubbles. To do so, place the tip of the pipette onthe side of the tube. The tube portion (0.12 ml) is filled on all thetubes except tube 50 where the tube and cup section are filled so thatthe reading can be performed in the cup (aerobic conditions). Two dropsof sterile mineral oil are added to all the tubes underlined (0, 1, 2,etc.)

The plastic box containing the inoculated plate is closed and placed inthe incubator. If the incubator does not circulate humid atmosphere, thebox should be placed on one of the top shelves far from the heatingunits. The incubation is performed at 37°C except for the L.viridenscens when a temperature of 30°C is recommended.

In forming a profile, the results can be compiled on the result sheet51. When the reaction is considered as positive, it is reported on theresult sheet by blackening only the positive reactions corresponding tothe time of appearance (See FIG. 10.).

TESTS 1 THROUGH 40 -- FERMENTATION AND GROWTH

The acid production precedes the growth and is easier to read during thefirst 6 hour period. The changing from purple to yellow of thebromcresol purple is reported after 3 and 6 hours of incubation.

After 24 hours of incubation, the bacterial growth is also reported byobserving an opaque deposit at the bottom of the tube when the platesare observed by transparence on a black background. This reactionappears in addition to the fermentation. With some species such as theTHERMOBACTERIUM the bacterial growth is sometimes easier to read thanthe fermentation. Gas Production: The heterofermentative species areproducing gas which forms small bubbles on the upper part of the tubefrom 6 to 24 hours of incubation. Some species produce more gas with themaltose than with the glucose and the gas production may be observedwith other carbohydrates during fermentation.

The gas production will be read and reported in tube 42 (glucose) only.

The readings of the results after 3, 6 and 24 hours should be sufficientto enable the diagnosis. It is useless to report the results of thefermentation after 48 hours of incubation.

POSITIVE REACTION: YELLOW and GROWTH of the organism

NEGATIVE REACTION: PURPLE and NO GROWTH of the organism

The 50 biochemical reactions have been selected to enable the diagnosisof the group, the species and recognition of a strain from its profile.

                  DIAGNOSIS OF GROUPS                                             ______________________________________                                                    THERMO-    STREPTO-   BETA-                                       TEST        BACTERIUM  BACTERIUM  BACTERIUM                                   ______________________________________                                        RIBOSE      -          +          +                                           ARGININ     -          -          +                                           GAS PRODUCTION                                                                            -          -          +                                           IN GLUCOSE                                                                    ______________________________________                                    

Under each profile the differentiating characteristics which enable oneto diagnose the group are indicated by the following symbols:

BLACK CIRCLES 52 for the tests which are positive.

O WHITE CIRCLES 53 for the tests which are negative.

DIAGNOSIS OF THE SPECIES

Under each diagram the differentiating characteristics of the speciesare indicated by the following symbols:

BLACK TRIANGLES 54 for positive reactions

Δ WHITE TRIANGLES 55 for negative reactions

For example:

White circles under the ribose test arginin ADH and gas are apresumption of a THERMOBACTERIUM.

The black triangle under the maltose test on the L. helveticus profiledifferentiates this specie from the L. jugurti by the fermentation ofthis carbohydrate.

DIAGNOSIS OF A STRAIN

The profile of a strain must always remain identical to itself duringthe preparation, or at all times during the continuous culture. When theexperimental conditions are carefully reproduced, it is possible toreport a modification in the profile much faster than with conventionalmethods. The strain can be checked every hour or every two hours. As anexample, the follow-up of a continuous culture of a L. helvecticusstrain and its stopping because of a variation in the aspect of theprofile.

What is claimed is:
 1. In a method for the identification of an unknownsubstance, a specimen of the substance having been prepared to beanalyzed, portions of the prepared specimen being caused to undergo aplurality of different tests, each test having a predetermined indiciawhen the specimen has not reacted and a different predetermined indiciawhen the specimen has reacted, the improvement which comprises:a.arranging the tests into at least one group which includes a pluralityof individual test determinations, each test determination beingassigned a score value, the score value for each determination beingdifferent depending upon whether the specimen has or has not reacted,the score value of each determination in a group being weighteddifferently for the condition when the specimen has reacted, and the sumof any combination of score values in a group being different from thesum of any other combination of score values within the group; b.comparing the predetermined indicia and the different predeterminedindicia of each test to an apparatus having a supporting frame and atleast one member corresponding to a group of determinations andcontaining a representation for the condition when the specimen has notreacted and a representation for the condition when the specimen hasreacted, each member being divided into fixed increments, the magnitudeof each increment being a function of the weighted score value for eachdetermination in a group corresponding to the movable member, whereinsequential movements of the movable member provide a total movementrepresentative of the combining of score values of the group and thecorrect indicia of reaction for each determination in a group; c. inresponse to the step of comparing, moving each movable member throughthe increment for each determination in a group to which the movablemember is related when the specimen has reacted, the moving of themovable member both exposing the representation for the condition whenthe specimen has reacted and forming a score value for eachdetermination, the movable member being moved sequentially until a totalgroup score value for each group is obtained, the representation of eachmovable member after moving enabling the representation to be checkedagainst the predetermined indicia and different predetermined indicia ofthe test; and d. assembling the score values of all groups into a finalvalue which is distinctive for each different combination of group scorevalues, the final value being indicative of the identification of theunknown substance.
 2. In a method in accordance with claim 1 in whichthe step of arranging the tests into at least one group comprisesarranging the tests into groups containing equal numbers ofdeterminations.
 3. In a method in accordance with claim 1 in which eachgroup includes three determinations.
 4. In a method in accordance withclaim 1 in which each group includes five determinations.
 5. In a methodin accordance with claim 1 in which the score value comprises anumerical score value.
 6. In a method in accordance with claim 5 inwhich the numerical score value is zero for the condition in which thespecimen has not reacted and a numerical score value different from zerofor the condition in which the specimen has reacted.
 7. In a method inaccordance with claim 6 in which the numerical score value differentfrom zero for each different determination in a group is weighted withan increasing numerical value in a predetermined manner.
 8. In a methodin accordance with claim 7 in which the numerical score value which isweighted with an increasing numerical value in a predetermined manner isweighted as a function of powers of a predetermined base.
 9. In a methodin accordance with claim 8 in which the predetermined base is two. 10.In a method in accordance with claim 5 in which each group includesthree determinations and in which the numerical score value is zero forthe condition in which the specimen has not reacted and a numericalscore value of 1, 2, and 4 for the three determinations, respectively,for the condition in which the specimen has reacted.
 11. In a method inaccordance with claim 5 in which each group includes five determinationsand in which the numerical score value is zero for the condition inwhich the specimen has not reacted and a numerical score value of 1, 2,4, 8 and 16 for the five determinations, respectively, for the conditionin which the specimen has reacted.
 12. In a method in accordance withclaim 5 in which each numerical score value of a group is mathematicallycombined with a preceding recorded score value.
 13. In a method inaccordance with claim 12 in which the numerical score values aremathematically combined by summation of the numerical score values, theresulting sum being the group score value.
 14. In a method in accordancewith claim 1 in which the step of assembling the score values of allgroups into a final value comprises the step of placing the score valueof all groups into different positions in accordance with apredetermined format, the format presenting the final value.
 15. In amethod in accordance with claim 14 in which the step of positioning thescore values of all groups into a predetermined format comprisesarranging the score values in a sequence in correspondence to thepredetermined order of the groups.
 16. In a method in accordance withclaim 1 and further comprising the step of identifying the unknownsubstance by consulting a tabular register of possible final values. 17.In a method in accordance with claim 16, wherein the tabular registerincludes representations of the final values which are in at least oneof a plurality of different predetermined forms, each differentpredetermined form corresponding to a different predetermined conditionof the prepared specimen, and wherein the step of consulting the tabularregister includes the step of consulting the predetermined formcorresponding to the predetermined condition of the prepared specimen.18. In a method for the identification of an unknown micro-organism, aspecimen of the micro-organism having been prepared to be analyzed,portions of the prepared specimen being caused to undergo a plurality ofdifferent biochemical tests with different biochemical test reagentseach test having a predetermined indicia when the specimen has notreacted and a different predetermined indicia when the specimen hasreacted, at least certain of the reagents having a chemical reactionindicating substance, the improvement which comprises:a. arranging thetests into at least one group which includes a plurality of individualtest determinations, each test determination being assigned a scorevalue, the score value for each determination being different dependingupon whether the specimen has or has not been reacted, the score valueof each determination in a group being weighted differently for thecondition when the specimen has reacted, and the sum of any combinationof score values in a group being different from the sum of any othercombination of score values within the group; b. comparing thepredetermined indicia and the different predetermined indicia of eachtest to an apparatus having a supporting frame and at least one membercorresponding to a group of determinations and containing arepresentation for the condition when the specimen has not reacted, anda representation for the condition when the specimen has reacted eachmember being divided into fixed increments, the magnitude of eachincrement being a function of the weighted score value for eachdetermination in a group corresponding to the movable member, whereinsequential movements of the movable member provide a total movementrepresentative of the combining of score values of the group and thecorrect indicia of reaction for each determination in a group; c. inresponse to the step of comparing, moving each movable member throughthe increment for each determination in a group to which the movablemember is related when the specimen has reacted, the moving of themovable member both exposing the representation for the condition whenthe specimen has reacted and forming a score value for eachdetermination, the movable member being moved sequentially until a totalgroup score value for each group is obtained, the representation of eachmovable member after moving enabling the representation to be checkedagainst the predetermined indicia and different predetermined indicia ofthe test; and d. assembling the score values of all groups into a finalvalue which is distinctive for each different combination of group scorevalues, the final value being indicative of the indentification of theunknown substance.
 19. In a method for the identification of an unknownbacterium, a specimen of the bacterium having been prepared to beanalyzed, portions of the prepared specimen being caused to react with aplurality of tests with different biochemical test reagents each testhaving a predetermined indicia when the specimen has not reacted and adifferent predetermined indicia when the specimen has reacted, at leastcertain of the reagents having a chemical reaction indicating substance,the improvement which comprises:a. arranging the tests into at least onegroup which includes a plurality of individual test determinations, eachtest determination being assigned a score value, the score value foreach determination being different depending upon whether the specimenhas or has not reacted, the score value of each determination in a groupbeing weighted differently for the condition when the specimen hasreacted, and the sum of any combination of score values in a group beingdifferent from the sum of any other combination of score values withinthe group; b. comparing the predetermined indicia and the differentpredetermined indicia of each test to an apparatus having a supportingframe and at least one member corresponding to a group of determinationsand containing a representation for the condition when the specimen hasnot reacted and a representation for the condition when the specimen hasreacted, each member being divided into fixed increments, the magnitudeof each increment being a function of the weighted score value for eachdetermination in a group corresponding to the movable member, whereinsequential movements of the movable member provide a total movementrepresentative of the combining of score values of the group and thecorrect indicia of reaction for each determination in a group; c. inresponse to the step of comparing, moving each movable member throughthe increment for each determination in a group to which the movablemember is related when the specimen has reacted, the moving of themovable member both exposing the representation for the condition whenthe specimen has reacted and forming a score value for eachdetermination, the movable member being moved sequentially until a totalgroup score value for each group is obtained, the representation of eachmovable member after moving enabling the representation to be checkedagainst the predetermined indicia and different predetermined indicia ofthe test; and d. assembling the score values of all groups into a finalvalue which is distinctive for each different combination of group scorevalues, the final value being indicative of the indentification of theunknown substance.
 20. In a method in accordance with claim 19 in whichthe step of arranging the tests into at least one group comprisesarranging the tests into groups containing equal numbers ofdeterminations.
 21. In a method in accordance with claim 20 in whicheach group includes five determinations.
 22. In a method in accordancewith claim 19 in which the step of assembling the numerical score valuesof all groups into a final value comprises the step of placing thenumerical score values of all groups into different positions inaccordance with a predetermined format. The format presenting the finalvalue.
 23. In a method in accordance with claim 22 in which the step ofpositioning the numerical group score values of all groups into apredetermined format comprises arranging the numerical group scorevalues into a sequence in correspondence to the predetermined order ofthe groups.